Chapter 27 Amino Acids, Peptides, and Proteins Dr. Wolf's CHM 424 4

27.1 Classification of Amino Acids Dr. Wolf's CHM 424 4

Fundamentals While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively. They are classified as , , , etc. amino acids according the carbon that bears the nitrogen. Dr. Wolf's CHM 424

Amino Acids + NH3  – CO2 + – H3NCH2CH2CO2  + – H3NCH2CH2CH2CO2  an -amino acid that is an intermediate in the biosynthesis of ethylene  a -amino acid that is one of the structural units present in coenzyme A + H3NCH2CH2CO2 –  + H3NCH2CH2CH2CO2 – a -amino acid involved in the transmission of nerve impulses  Dr. Wolf's CHM 424

They differ in respect to the group attached to the  carbon. The 20 Key Amino Acids More than 700 amino acids occur naturally, but 20 of them are especially important. These 20 amino acids are the building blocks of proteins. All are -amino acids. They differ in respect to the group attached to the  carbon. These 20 are listed in Table 27.1. Dr. Wolf's CHM 424

The properties of the amino acid vary as the structure of R varies. Table 27.1 C O – R H H3N + The amino acids obtained by hydrolysis of proteins differ in respect to R (the side chain). The properties of the amino acid vary as the structure of R varies. Dr. Wolf's CHM 424

The major differences among the side chains concern: Table 27.1 C O – R H H3N + The major differences among the side chains concern: Size and shape Electronic characteristics Dr. Wolf's CHM 424

General categories of a-amino acids Table 27.1 General categories of a-amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Dr. Wolf's CHM 424

General categories of a-amino acids Table 27.1 General categories of a-amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Dr. Wolf's CHM 424

Table 27.1 C O – H H3N + Glycine (Gly or G) Glycine is the simplest amino acid. It is the only one in the table that is achiral. In all of the other amino acids in the table the  carbon is a chirality center. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O CH3 Alanine (Ala or A) Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O CH(CH3)2 Valine (Val or V) Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O CH2CH(CH3)2 Leucine (Leu or L) Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C CH3CHCH2CH3 Isoleucine (Ile or I) Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O CH3SCH2CH2 Methionine (Met or M) The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable. Dr. Wolf's CHM 424

Table 27.1 H O + – H2N C Proline H2C CH2 (Pro or P) C H2 Proline is the only amino acid that contains a secondary amine function. Its side chain is nonpolar and cyclic. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C CH2 Phenylalanine (Phe or F) The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C Tryptophan CH2 (Trp or W) The side chain in tryptophan (a nonpolar amino acid) is larger and more polarizable than the benzyl group of phenylalanine. Dr. Wolf's CHM 424

General categories of a-amino acids Table 27.1 General categories of a-amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Dr. Wolf's CHM 424

The —CH2OH side chain in serine can be involved in hydrogen bonding. Table 27.1 H O + – H3N C C O CH2OH Serine (Ser or S) The —CH2OH side chain in serine can be involved in hydrogen bonding. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O CH3CHOH Threonine (Thr or T) The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O CH2SH Cysteine (Cys or C) The side chains of two remote cysteines can be joined by forming a covalent S—S bond. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C Tyrosine CH2 (Tyr or Y) The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C Asparagine H2NCCH2 (Asn or N) The side chains of asparagine and glutamine (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C Glutamine H2NCCH2CH2 (Gln or Q) Dr. Wolf's CHM 424

General categories of a-amino acids Table 27.1 General categories of a-amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C Aspartic Acid OCCH2 (Asp or D) Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They are negatively charged and can form ionic bonds with positively charged species. Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C Glutamic Acid OCCH2CH2 (Glu or E) Dr. Wolf's CHM 424

General categories of a-amino acids Table 27.1 General categories of a-amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O Lysine + (Lys or K) CH2CH2CH2CH2NH3 Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are positively charged and can form ionic bonds with negatively charged species. Dr. Wolf's CHM 424

Table 27.1 H O + – Arginine H3N C C O (Arg or R) CH2CH2CH2NHCNH2 + NH2 Dr. Wolf's CHM 424

Table 27.1 H O + – H3N C C O Histidine (His or H) Histidine is a basic amino acid, but less basic than lysine and arginine. Histidine can interact with metal ions and can help move protons from one site to another. CH2 NH N Dr. Wolf's CHM 424

27.2 Stereochemistry of Amino Acids Dr. Wolf's CHM 424 4

Configuration of -Amino Acids Glycine is achiral. All of the other amino acids in proteins have the L-configuration at their carbon. H3N + H R CO2 – Dr. Wolf's CHM 424

27.3 Acid-Base Behavior of Amino Acids Dr. Wolf's CHM 424 4

Recall While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively. How do we know this? Dr. Wolf's CHM 424

The properties of glycine: high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent O OH H2NCH2C •• • • – H3NCH2C + more consistent with this than this Dr. Wolf's CHM 424

called a zwitterion or dipolar ion Properties of Glycine The properties of glycine: high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent more consistent with this called a zwitterion or dipolar ion – • • O H3NCH2C •• + Dr. Wolf's CHM 424

Acid-Base Properties of Glycine The zwitterionic structure of glycine also follows from considering its acid-base properties. A good way to think about this is to start with the structure of glycine in strongly acidic solution, say pH = 1. At pH = 1, glycine exists in its protonated form (a monocation). O OH H3NCH2C + •• • • Dr. Wolf's CHM 424

Acid-Base Properties of Glycine Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is it the proton of the carboxyl group?" You can choose between them by estimating their respective pKas. typical ammonium ion: pKa ~9 typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • • Dr. Wolf's CHM 424

Acid-Base Properties of Glycine The more acidic proton belongs to the CO2H group. It is the first one removed as the pH is raised. typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • • Dr. Wolf's CHM 424

Acid-Base Properties of Glycine Therefore, the more stable neutral form of glycine is the zwitterion. – • • O H3NCH2C •• + typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • • Dr. Wolf's CHM 424

Acid-Base Properties of Glycine The measured pKa of glycine is 2.34. Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an electron-withdrawing, acid-strengthening substituent on the  carbon. typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • • Dr. Wolf's CHM 424

Acid-Base Properties of Glycine A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further. – • • O H3NCH2C •• + – • • O H2NCH2C •• HO – The pKa for removal of this proton is 9.60. This value is about the same as that for NH4+ (9.3). Dr. Wolf's CHM 424

Isoelectric Point pI O OH H3NCH2C + •• • • The pH at which the concentration of the zwitterion is a maximum is called the isoelectric point. Its numerical value is the average of the two pKas. The pI of glycine is 5.97. pKa = 2.34 – • • O H3NCH2C •• + pKa = 9.60 – • • O H2NCH2C •• Dr. Wolf's CHM 424

Acid-Base Properties of Amino Acids One way in which amino acids differ is in respect to their acid-base properties. This is the basis for certain experimental methods for separating and identifying them. Just as important, the difference in acid-base properties among various side chains affects the properties of the proteins that contain them. Table 27.2 gives pKa and pI values for amino acids with neutral side chains. Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – H H3N + pKa1 = 2.34 pKa2 = 9.60 pI = 5.97 Glycine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – CH3 H + pKa1 = 2.34 pKa2 = 9.69 pI = 6.00 Alanine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – CH(CH3)2 H + pKa1 = 2.32 pKa2 = 9.62 pI = 5.96 Valine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – CH2CH(CH3)2 H + pKa1 = 2.36 pKa2 = 9.60 pI = 5.98 Leucine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains pKa1 = 2.36 pKa2 = 9.60 pI = 5.98 + – Isoleucine H3N C C O CH3CHCH2CH3 Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains pKa1 = 2.28 pKa2 = 9.21 pI = 5.74 + – Methionine H3N C C O CH3SCH2CH2 Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – H + CH2 H2C C H2 pKa1 = 1.99 pKa2 = 10.60 pI = 6.30 Proline Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – H + CH2 pKa1 = 1.83 pKa2 = 9.13 pI = 5.48 Phenylalanine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – H + CH2 N pKa1 = 2.83 pKa2 = 9.39 pI = 5.89 Tryptophan Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains pKa1 = 2.02 pKa2 = 8.80 pI = 5.41 + – Asparagine H3N C C O H2NCCH2 O Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – H + H2NCCH2CH2 pKa1 = 2.17 pKa2 = 9.13 pI = 5.65 Glutamine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – CH2OH H + pKa1 = 2.21 pKa2 = 9.15 pI = 5.68 Serine Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains pKa1 = 2.09 pKa2 = 9.10 pI = 5.60 + – Threonine H3N C C O CH3CHOH Dr. Wolf's CHM 424

Table 27.2 Amino Acids with Neutral Side Chains – H + CH2 OH pKa1 = 2.20 pKa2 = 9.11 pI = 5.66 Tyrosine Dr. Wolf's CHM 424

Table 27.3 Amino Acids with Neutral Side Chains – CH2SH H + pKa1 = 1.96 pKa2 = 8.18 pI = 5.07 Cysteine Dr. Wolf's CHM 424

Table 27.3 Amino Acids with Ionizable Side Chains pKa1 = 1.88 pKa2 = 3.65 pKa3 = 9.60 pI = 2.77 + – Aspartic acid H3N C C O OCCH2 O – For amino acids with acidic side chains, pI is the average of pKa1 and pKa2. Dr. Wolf's CHM 424

Table 27.3 Amino Acids with Ionizable Side Chains pKa1 = 2.19 pKa2 = 4.25 pKa3 = 9.67 pI = 3.22 + – Glutamic acid H3N C C O OCCH2CH2 – O Dr. Wolf's CHM 424

Table 27.3 Amino Acids with Ionizable Side Chains H3N C O – H + CH2CH2CH2CH2NH3 pKa1 = 2.18 pKa2 = 8.95 pKa3 = 10.53 pI = 9.74 Lysine For amino acids with basic side chains, pI is the average of pKa2 and pKa3. Dr. Wolf's CHM 424

Table 27.3 Amino Acids with Ionizable Side Chains H3N C O – H + CH2CH2CH2NHCNH2 NH2 pKa1 = 2.17 pKa2 = 9.04 pKa3 = 12.48 pI = 10.76 Arginine Dr. Wolf's CHM 424

Table 27.3 Amino Acids with Ionizable Side Chains H3N C O – H + CH2 NH N pKa1 = 1.82 pKa2 = 6.00 pKa3 = 9.17 pI = 7.59 Histidine Dr. Wolf's CHM 424

27.4 Synthesis of Amino Acids Dr. Wolf's CHM 424 4

From -Halo Carboxylic Acids CH3CHCOH Br O CH3CHCO NH3 O + – (65-70%) NH4Br H2O + 2NH3 Dr. Wolf's CHM 424

Strecker Synthesis CH3CH O NH4Cl CH3CHC NH2 N NaCN CH3CHCO NH3 O + – (52-60%) 1. H2O, HCl, heat 2. HO– Dr. Wolf's CHM 424

Using Diethyl Acetamidomalonate OCH2CH3 H O CH3CH2O CH3CNH Can be used in the same manner as diethyl malonate (Section 21.7). Dr. Wolf's CHM 424

Example O CH3CH2OCCCOCH2CH3 CH3CNH H O 1. NaOCH2CH3 2. C6H5CH2Cl O (90%) O Dr. Wolf's CHM 424

O O Example HOCCCOH CH2C6H5 H3N + –CO2 O HBr, H2O, heat HCCOH CH2C6H5 (65%) –CO2 HBr, H2O, heat O CH3CH2OCCCOCH2CH3 CH2C6H5 CH3CNH Dr. Wolf's CHM 424

27.5 Reactions of Amino Acids Dr. Wolf's CHM 424 4

Acylation of Amino Group The amino nitrogen of an amino acid can be converted to an amide with the customary acylating agents. O CH3COCCH3 O H3NCH2CO – + + CH3CNHCH2COH O (89-92%) Dr. Wolf's CHM 424

Esterification of Carboxyl Group The carboxyl group of an amino acid can be converted to an ester. The following illustrates Fischer esterification of alanine. O H3NCHCO – + CH3 + CH3CH2OH HCl (90-95%) O H3NCHCOCH2CH3 + CH3 – Cl Dr. Wolf's CHM 424

Ninhydrin Test OH O O H3NCHCO – + R + O N – O RCH + CO2 + H2O + Amino acids are detected by the formation of a purple color on treatment with ninhydrin. OH O O H3NCHCO – + R + O N – O RCH + CO2 + H2O + Dr. Wolf's CHM 424

27.6 Some Biochemical Reactions of Amino Acids Dr. Wolf's CHM 424 4

Biosynthesis of L-Glutamic Acid HO2CCH2CH2CCO2H O + NH3 enzymes and reducing coenzymes HO2CCH2CH2CHCO2 – NH3 + This reaction is the biochemical analog of reductive amination (Section 22.10). Dr. Wolf's CHM 424

Transamination via L-Glutamic Acid HO2CCH2CH2CHCO2 – NH3 + CH3CCO2H O L-Glutamic acid acts as a source of the amine group in the biochemical conversion of -keto acids to other amino acids. In the example to be shown, pyruvic acid is converted to L-alanine. Dr. Wolf's CHM 424

Transamination via L-Glutamic Acid HO2CCH2CH2CHCO2 – NH3 + CH3CCO2H O enzymes HO2CCH2CH2CCO2H O CH3CHCO2 – NH3 + + Dr. Wolf's CHM 424

Mechanism O – HO2CCH2CH2CHCO2 CH3CCO2H + NH3 The first step is imine formation between the amino group of L-glutamic acid and pyruvic acid. Dr. Wolf's CHM 424

Mechanism O – HO2CCH2CH2CHCO2 CH3CCO2H + NH3 – HO2CCH2CH2CHCO2 N – Dr. Wolf's CHM 424

Formation of the imine is followed by proton removal at one carbon and protonation of another carbon. H CH3CCO2 – HO2CCH2CH2CCO2 N Dr. Wolf's CHM 424

– HO2CCH2CH2CCO2 N – CH3CCO2 H H HO2CCH2CH2CCO2 N – CH3CCO2 Dr. Wolf's CHM 424

Hydrolysis of the imine function gives -keto glutarate and L-alanine. – CH3CCO2 – HO2CCH2CH2CCO2 N H Hydrolysis of the imine function gives -keto glutarate and L-alanine. Dr. Wolf's CHM 424

– HO2CCH2CH2CCO2 N – CH3CCO2 H H2O + NH3 – – HO2CCH2CH2CCO2 + CH3CCO2 Dr. Wolf's CHM 424

Biosynthesis of L-Tyrosine L-Tyrosine is biosynthesized from L-phenylalanine. A key step is epoxidation of the aromatic ring to give an arene oxide intermediate. CH2CHCO2 – NH3 + Dr. Wolf's CHM 424

Biosynthesis of L-Tyrosine CH2CHCO2 – NH3 + O O2, enzyme CH2CHCO2 – NH3 + Dr. Wolf's CHM 424

Biosynthesis of L-Tyrosine CH2CHCO2 – NH3 + O enzyme CH2CHCO2 – NH3 + HO Dr. Wolf's CHM 424

Biosynthesis of L-Tyrosine Conversion to L-tyrosine is one of the major metabolic pathways of L-phenylalanine. Individuals who lack the enzymes necessary to convert L-phenylalanine to L-tyrosine can suffer from PKU disease. In PKU disease, L-phenylalanine is diverted to a pathway leading to phenylpyruvic acid, which is toxic. Newborns are routinely tested for PKU disease. Treatment consists of reducing their dietary intake of phenylalanine-rich proteins. Dr. Wolf's CHM 424

Decarboxylation Decarboxylation is a common reaction of -amino acids. An example is the conversion of L-histidine to histamine. Antihistamines act by blocking the action of histamine. CH2CHCO2 – NH3 + N H N Dr. Wolf's CHM 424

Decarboxylation N CH2CH2 NH2 N H –CO2, enzymes N – CH2CHCO2 N H + NH3 Dr. Wolf's CHM 424

Neurotransmitters – CO2 OH CO2 – H H3N + The chemistry of the brain and central nervous system is affected by neurotransmitters. Several important neurotransmitters are biosynthesized from L-tyrosine. L-Tyrosine Dr. Wolf's CHM 424

Neurotransmitters – CO2 The common name of this compound is L-DOPA. It occurs naturally in the brain. It is widely prescribed to reduce the symptoms of Parkinsonism. + H3N H H H HO OH L-3,4-Dihydroxyphenylalanine Dr. Wolf's CHM 424

Dopamine is formed by decarboxylation of L-DOPA. H2N H H H Neurotransmitters H Dopamine is formed by decarboxylation of L-DOPA. H2N H H H HO OH Dopamine Dr. Wolf's CHM 424

Neurotransmitters H H2N H H OH HO OH Norepinephrine Dr. Wolf's CHM 424

Neurotransmitters H CH3NH H H OH HO OH Epinephrine Dr. Wolf's CHM 424

27.7 Peptides Dr. Wolf's CHM 424 4

An amide bond of this type is often referred to as a peptide bond. Peptides Peptides are compounds in which an amide bond links the amino group of one -amino acid and the carboxyl group of another. An amide bond of this type is often referred to as a peptide bond. Dr. Wolf's CHM 424

Alanine and Glycine CH3 O C + H – H3N O C H H3N + – Dr. Wolf's CHM 424

Alanylglycine CH3 O C H3N + H N – Two -amino acids are joined by a peptide bond in alanylglycine. It is a dipeptide. Dr. Wolf's CHM 424

Alanylglycine CH3 O C H3N + H N – N-terminus C-terminus Ala—Gly AG Dr. Wolf's CHM 424

Alanylglycine and glycylalanine are constitutional isomers CH3 O C H3N + H N – Alanylglycine Ala—Gly AG H O C H3N + N CH3 – Glycylalanine Gly—Ala GA Dr. Wolf's CHM 424

The peptide bond is characterized by a planar geometry. Alanylglycine CH3 O C H3N + H N – The peptide bond is characterized by a planar geometry. Dr. Wolf's CHM 424

dipeptides, tripeptides, tetrapeptides, etc. Higher Peptides Peptides are classified according to the number of amino acids linked together. dipeptides, tripeptides, tetrapeptides, etc. Leucine enkephalin is an example of a pentapeptide. Dr. Wolf's CHM 424

Tyr—Gly—Gly—Phe—Leu YGGFL Leucine Enkephalin Tyr—Gly—Gly—Phe—Leu YGGFL Dr. Wolf's CHM 424

Oxytocin is a cyclic nonapeptide. Ile—Gln—Asn Tyr Cys S Cys—Pro—Leu—GlyNH2 1 2 3 4 5 6 7 8 9 C-terminus N-terminus Oxytocin is a cyclic nonapeptide. Instead of having its amino acids linked in an extended chain, two cysteine residues are joined by an S—S bond. Dr. Wolf's CHM 424

Oxytocin S—S bond An S—S bond between two cysteines is often referred to as a disulfide bridge. Dr. Wolf's CHM 424

27.8 Introduction to Peptide Structure Determination Dr. Wolf's CHM 424 4

Primary Structure The primary structure is the amino acid sequence plus any disulfide links. Dr. Wolf's CHM 424

Classical Strategy (Sanger) 1. Determine what amino acids are present and their molar ratios. 2. Cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragments. 3. Identify the N-terminus and C-terminus in the parent peptide and in each fragment. 4. Organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence. Dr. Wolf's CHM 424

27.9 Amino Acid Analysis Dr. Wolf's CHM 424 4

Amino acids are detected using ninhydrin. Amino Acid Analysis Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids. The mixture is separated by ion-exchange chromatography, which depends on the differences in pI among the various amino acids. Amino acids are detected using ninhydrin. Automated method; requires only 10-5 to 10-7 g of peptide. Dr. Wolf's CHM 424

27.10 Partial Hydrolysis of Proteins Dr. Wolf's CHM 424 4

Partial Hydrolysis of Peptides and Proteins Acid-hydrolysis of the peptide cleaves all of the peptide bonds. Cleaving some, but not all, of the peptide bonds gives smaller fragments. These smaller fragments are then separated and the amino acids present in each fragment determined. Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis. Dr. Wolf's CHM 424

Partial Hydrolysis of Peptides and Proteins The enzymes that catalyze the hydrolysis of peptide bonds are called peptidases, proteases, or proteolytic enzymes. Dr. Wolf's CHM 424

Trypsin Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine. lysine or arginine NHCHC O R' R" R Dr. Wolf's CHM 424

phenylalanine, tyrosine, tryptophan NHCHC O Chymotrypsin Chymotrypsin is selective for cleaving the peptide bond to the carboxyl group of amino acids with an aromatic side chain. phenylalanine, tyrosine, tryptophan NHCHC O R' R" R Dr. Wolf's CHM 424

Carboxypeptidase Carboxypeptidase is selective for cleaving the peptide bond to the C-terminal amino acid. protein H3NCHC O R + NHCHCO – C Dr. Wolf's CHM 424

27.11 End Group Analysis Dr. Wolf's CHM 424 4

We need to know what the N-terminal and C-terminal amino acids are. End Group Analysis Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left. We need to know what the N-terminal and C-terminal amino acids are. The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis. Several chemical methods have been developed for identifying the N-terminus. They depend on the fact that the amino N at the terminus is more nucleophilic than any of the amide nitrogens. Dr. Wolf's CHM 424

Sanger's Method The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene. 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution (Section 23.5). F O2N NO2 Dr. Wolf's CHM 424

Sanger's Method 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. F O2N NO2 NHCH2C NHCHCO CH3 NHCHC CH2C6H5 H2NCHC O CH(CH3)2 – + O2N NO2 NHCH2C NHCHCO CH3 NHCHC CH2C6H5 O CH(CH3)2 – Dr. Wolf's CHM 424

Sanger's Method Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group. H3O+ O O2N NO2 NHCHCOH CH(CH3)2 H3NCHCO– CH3 + H3NCH2CO– O CH2C6H5 O2N NO2 NHCH2C NHCHCO CH3 NHCHC CH2C6H5 O CH(CH3)2 – Dr. Wolf's CHM 424

27.12 Insulin Dr. Wolf's CHM 424 4

Insulin is a polypeptide with 51 amino acids. It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids). The following describes how the amino acid sequence of the B chain was determined. Dr. Wolf's CHM 424

The B Chain of Bovine Insulin Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Dr. Wolf's CHM 424

The B Chain of Bovine Insulin FVNQHLCGSHL VGAL VCGERGF YTPKA Dr. Wolf's CHM 424

The B Chain of Bovine Insulin Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC Dr. Wolf's CHM 424

The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALT TLVC VCGERGF YTPKA Dr. Wolf's CHM 424

The B Chain of Bovine Insulin Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence. Dr. Wolf's CHM 424

The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALT TLVC VCGERGF GFFYTPK YTPKA Dr. Wolf's CHM 424

The B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALT TLVC VCGERGF GFFYTPK YTPKA FVNQHLCGSHLVGALTLVCGERGFFYTPKA Dr. Wolf's CHM 424

The sequence of the A chain was determined using the same strategy. Insulin The sequence of the A chain was determined using the same strategy. Establishing the disulfide links between cysteine residues completed the primary structure. Dr. Wolf's CHM 424

Primary Structure of Bovine Insulin N terminus of A chain 5 15 10 20 25 30 S F V N Q H L C G A Y E R I K P T C terminus of A chain N terminus of B chain C terminus of B chain Dr. Wolf's CHM 424

27.13 The Edman Degradation and Automated Sequencing of Peptides Dr. Wolf's CHM 424 4

1. Method for determining N-terminal amino acid. Edman Degradation 1. Method for determining N-terminal amino acid. 2. Can be done sequentially one residue at a time on the same sample. Usually one can determine the first 20 or so amino acids from the N-terminus by this method. 3. 10-10 g of sample is sufficient. 4. Has been automated. Dr. Wolf's CHM 424

The key reagent in the Edman degradation is phenyl isothiocyanate. Dr. Wolf's CHM 424

Edman Degradation Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid. peptide H3NCHC O R + NH C6H5N C S + Dr. Wolf's CHM 424

Edman Degradation peptide C6H5NHCNHCHC O R NH S peptide H3NCHC O R + Dr. Wolf's CHM 424

The product is a phenylthiocarbamoyl (PTC) derivative. Edman Degradation peptide C6H5NHCNHCHC O R NH S The product is a phenylthiocarbamoyl (PTC) derivative. The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide. Dr. Wolf's CHM 424

Edman Degradation peptide C6H5NHCNHCHC O R NH S HCl C6H5NH C S N CH R + + Dr. Wolf's CHM 424

The product is a thiazolone. Under the Edman Degradation The product is a thiazolone. Under the conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH) derivative. C6H5NH C S N CH R O peptide H3N + + Dr. Wolf's CHM 424

Edman Degradation C N HN CH R O S C6H5 The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation. C6H5NH C S N CH R O peptide H3N + + Dr. Wolf's CHM 424

27.14 The Strategy of Peptide Synthesis Dr. Wolf's CHM 424 4

General Considerations Making peptide bonds between amino acids is not difficult. The challenge is connecting amino acids in the correct sequence. Random peptide bond formation in a mixture of phenylalanine and glycine, for example, will give four dipeptides. Phe—Phe Gly—Gly Phe—Gly Gly—Phe Dr. Wolf's CHM 424

N-Protected phenylalanine General Strategy 1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other. N-Protected phenylalanine C-Protected glycine NHCHCOH CH2C6H5 O X H2NCH2C Y Dr. Wolf's CHM 424

2. Couple the two protected amino acids. General Strategy 2. Couple the two protected amino acids. NHCH2C O Y NHCHC CH2C6H5 X NHCHCOH CH2C6H5 O X H2NCH2C O Y Dr. Wolf's CHM 424

General Strategy 3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus. NHCH2C O Y NHCHC CH2C6H5 X NHCH2CO O H3NCHC CH2C6H5 + – Phe-Gly Dr. Wolf's CHM 424

27.15 Amino Group Protection Dr. Wolf's CHM 424 4

Protect Amino Groups as Amides Amino groups are normally protected by converting them to amides. Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z. Z-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride. Dr. Wolf's CHM 424

Protect Amino Groups as Amides CH2OCCl O H3NCHCO CH2C6H5 O – + + 1. NaOH, H2O 2. H+ NHCHCOH CH2C6H5 O CH2OC (82-87%) Dr. Wolf's CHM 424

Protect Amino Groups as Amides NHCHCOH CH2C6H5 O CH2OC is abbreviated as: ZNHCHCOH CH2C6H5 O or Z-Phe Dr. Wolf's CHM 424

Removing Z-Protection An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by: a) hydrogenolysis b) cleavage with HBr in acetic acid Dr. Wolf's CHM 424

Hydrogenolysis of Z-Protecting Group NHCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2OC H2, Pd H2NCHCNHCH2CO2CH2CH3 CH2C6H5 O CH3 CO2 (100%) Dr. Wolf's CHM 424

HBr Cleavage of Z-Protecting Group NHCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2OC HBr H3NCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2Br CO2 (82%) + Br – Dr. Wolf's CHM 424

The tert-Butoxycarbonyl Protecting Group NHCHCOH CH2C6H5 O (CH3)3COC is abbreviated as: BocNHCHCOH CH2C6H5 O or Boc-Phe Dr. Wolf's CHM 424

HBr Cleavage of Boc-Protecting Group (CH3)3COC NHCHCNHCH2CO2CH2CH3 CH2C6H5 HBr H3NCHCNHCH2CO2CH2CH3 CH2C6H5 O CO2 (86%) + Br – CH2 C H3C Dr. Wolf's CHM 424

27.16 Carboxyl Group Protection Dr. Wolf's CHM 424 4

Protect Carboxyl Groups as Esters Carboxyl groups are normally protected as esters. Deprotection of methyl and ethyl esters is by hydrolysis in base. Benzyl esters can be cleaved by hydrogenolysis. Dr. Wolf's CHM 424

Hydrogenolysis of Benzyl Esters NHCHCNHCH2COCH2C6H5 CH2C6H5 O C6H5CH2OC H2, Pd H3NCHCNHCH2CO CH2C6H5 O C6H5CH3 CO2 (87%) + – CH3C6H5 Dr. Wolf's CHM 424

27.17 Peptide Bond Formation Dr. Wolf's CHM 424 4

The two major methods are: Forming Peptide Bonds The two major methods are: 1. coupling of suitably protected amino acids using N,N'-dicyclohexylcarbodiimide (DCCI) 2. via an active ester of the N-terminal amino acid. Dr. Wolf's CHM 424

DCCI-Promoted Coupling ZNHCHCOH CH2C6H5 O H2NCH2COCH2CH3 O + DCCI, chloroform ZNHCHC CH2C6H5 O NHCH2COCH2CH3 (83%) Dr. Wolf's CHM 424

Mechanism of DCCI-Promoted Coupling ZNHCHCOH CH2C6H5 O + C6H11N C NC6H11 CH2C6H5 O C6H11N C H OCCHNHZ Dr. Wolf's CHM 424

Mechanism of DCCI-Promoted Coupling The species formed by addition of the Z-protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent. Attack by the amine function of the carboxyl-protected amino acid on the carbonyl group leads to nucleophilic acyl substitution. CH2C6H5 O C6H11N C H OCCHNHZ Dr. Wolf's CHM 424

Mechanism of DCCI-Promoted Coupling C6H11N C C6H11NH H O + ZNHCHC CH2C6H5 NHCH2COCH2CH3 H2NCH2COCH2CH3 O CH2C6H5 O C6H11N C H OCCHNHZ Dr. Wolf's CHM 424

The Active Ester Method A p-nitrophenyl ester is an example of an "active ester." p-Nitrophenyl is a better leaving group than methyl or ethyl, and p-nitrophenyl esters are more reactive in nucleophilic acyl substitution. Dr. Wolf's CHM 424

The Active Ester Method ZNHCHCO CH2C6H5 O H2NCH2COCH2CH3 O NO2 + chloroform ZNHCHC CH2C6H5 O NHCH2COCH2CH3 (78%) + HO NO2 Dr. Wolf's CHM 424

27.18 Solid-Phase Peptide Synthesis: The Merrifield Method Dr. Wolf's CHM 424 4

Solid-Phase Peptide Synthesis In solid-phase synthesis, the starting material is bonded to an inert solid support. Reactants are added in solution. Reaction occurs at the interface between the solid and the solution. Because the starting material is bonded to the solid, any product from the starting material remains bonded as well. Purification involves simply washing the byproducts from the solid support. Dr. Wolf's CHM 424

The Solid Support CH2 CH The solid support is a copolymer of styrene and divinylbenzene. It is represented above as if it were polystyrene. Cross-linking with divinylbenzene simply provides a more rigid polymer. Dr. Wolf's CHM 424

The Solid Support CH2 CH Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings. Dr. Wolf's CHM 424

The Solid Support CH2 CH CH2Cl The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2). Dr. Wolf's CHM 424

The Solid Support CH2 CH CH2Cl The chloromethylated resin is treated with the Boc-protected C-terminal amino acid. Nucleophilic substitution occurs, and the Boc-protected amino acid is bound to the resin as an ester. Dr. Wolf's CHM 424

The Merrifield Procedure CH2 CH CH2Cl BocNHCHCO R O – Dr. Wolf's CHM 424

The Merrifield Procedure CH2 CH BocNHCHCO R O Next, the Boc protecting group is removed with HCl. Dr. Wolf's CHM 424

The Merrifield Procedure H2NCHCO R CH2 CH O DCCI-promoted coupling adds the second amino acid Dr. Wolf's CHM 424

The Merrifield Procedure NHCHCO R O CH2 CH BocNHCHC R' Remove the Boc protecting group. Dr. Wolf's CHM 424

The Merrifield Procedure CH2 CH NHCHCO R O H2NCHC R' Add the next amino acid and repeat. Dr. Wolf's CHM 424

The Merrifield Procedure CH2 CH NHCHCO R O NHCHC R' C + H3N peptide Remove the peptide from the resin with HBr in CF3CO2H Dr. Wolf's CHM 424

The Merrifield Procedure CH2 CH CH2Br NHCHCO R O NHCHC R' C + H3N peptide – Dr. Wolf's CHM 424

Merrifield automated his solid-phase method. The Merrifield Method Merrifield automated his solid-phase method. Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield. Synthesized ribonuclease (124 amino acids) in 1969. 369 reactions; 11,391 steps Nobel Prize in chemistry: 1984 Dr. Wolf's CHM 424

27.19 Secondary Structures of Peptides and Proteins Dr. Wolf's CHM 424 4

Levels of Protein Structure Primary structure = the amino acid sequence plus disulfide links Secondary structure = conformational relationship between nearest neighbor amino acids  helix pleated  sheet Dr. Wolf's CHM 424

Levels of Protein Structure The -helix and pleated  sheet are both characterized by: planar geometry of peptide bond anti conformation of main chain hydrogen bonds between N—H and O=C Dr. Wolf's CHM 424

Pleated  Sheet Shown is a  sheet of protein chains composed of alternating glycine and alanine residues. Adjacent chains are antiparallel. Hydrogen bonds between chains. van der Waals forces produce pleated effect. Dr. Wolf's CHM 424

Pleated  Sheet  Sheet is most commonly seen with amino acids having small side chains (glycine, alanine, serine). 80% of fibroin (main protein in silk) is repeating sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.  Sheet is flexible, but resists stretching. Dr. Wolf's CHM 424

 Helix Shown is an  helix of a protein in which all of the amino acids are L-alanine. Helix is right-handed with 3.6 amino acids per turn. Hydrogen bonds are within a single chain. Protein of muscle (myosin) and wool (-keratin) contain large regions of -helix. Chain can be stretched. Dr. Wolf's CHM 424

27.20 Tertiary Structure of Peptides and Proteins Dr. Wolf's CHM 424 4

Refers to overall shape (how the chain is folded) Tertiary Structure Refers to overall shape (how the chain is folded) Fibrous proteins (hair, tendons, wool) have elongated shapes Globular proteins are approximately spherical most enzymes are globular proteins an example is carboxypeptidase Dr. Wolf's CHM 424

It is a metalloenzyme containing Zn2+ at its active site. Carboxypeptidase Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus. It is a metalloenzyme containing Zn2+ at its active site. An amino acid with a positively charged side chain (Arg-145) is near the active site. Dr. Wolf's CHM 424

Carboxypeptidase tube model ribbon model Disulfide bond Zn2+ Arg-145 N-terminus C-terminus tube model ribbon model Dr. Wolf's CHM 424

What happens at the active site? •• O O H2N • • + H3N peptide C NHCHC – + C Arg-145 R O H2N Dr. Wolf's CHM 424

What happens at the active site? •• O O H2N • • + H3N peptide C NHCHC – + C Arg-145 R O H2N The peptide or protein is bound at the active site by electrostatic attraction between its negatively charged carboxylate ion and arginine-145. Dr. Wolf's CHM 424

What happens at the active site? Zn2+ •• O O H2N • • + H3N peptide C NHCHC – + C Arg-145 R O H2N Zn2+ acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the carbonyl carbon. Dr. Wolf's CHM 424

What happens at the active site? Zn2+ •• O O H2N • • + H3N peptide C NHCHC – + C Arg-145 R O H2N O • • H Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs. Dr. Wolf's CHM 424

What happens at the active site? Zn2+ H2N + C Arg-145 H3N peptide O + C •• • • – H3NCHC R O – H2N + Dr. Wolf's CHM 424

27.21 Coenzymes Dr. Wolf's CHM 424 4

acid-base (transfer and accept protons) nucleophilic acyl substitution Coenzymes The range of chemical reactions that amino acid side chains can participate in is relatively limited. acid-base (transfer and accept protons) nucleophilic acyl substitution Many other biological processes, such as oxidation-reduction, require coenzymes, cofactors, or prosthetic groups in order to occur. Dr. Wolf's CHM 424

NADH, coenzyme A and coenzyme B12 are examples of coenzymes. Heme is another example. Dr. Wolf's CHM 424

Heme Molecule surrounding iron is a type of porphyrin. N Fe H3C CH3 CH2CH2CO2H CH CH2 H2C HO2CCH2CH2 Molecule surrounding iron is a type of porphyrin. Dr. Wolf's CHM 424

Myoglobin Heme N-terminus C-terminus Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen transport). Dr. Wolf's CHM 424

27.22 Protein Quaternary Structure: Hemoglobin Dr. Wolf's CHM 424 4

Protein Quaternary Structure Some proteins are assemblies of two or more chains. The way in which these chains are organized is called the quaternary structure. Hemoglobin, for example, consists of 4 subunits. There are 2  chains (identical) and 2  chains (also identical). Each subunit contains one heme and each protein is about the size of myoglobin. Dr. Wolf's CHM 424

End of Chapter 27 Dr. Wolf's CHM 424 4